Cotrimoxazole (Trimethoprim-sulfamethoxazole, TMP-SMX) is the agent of choice for prophylaxis against opportunistic infections associated with HIV/AIDS and other immuno-compromised states. However, hypersensitivity mediated by SMX limits its use. The goal of this project is to understand the genetic basis for variable sensitivity to SMX toxicity, an example of idiosyncratic adverse reactions observed with many drugs. Several pathways determine SMX hypersensitivity: Metabolic inactivation by N-acetyltransferases 1 and 2 (NAT1 and NAT2);metabolic oxidation to toxic hydroxylamine derivatives by CYP2C9 and other (extra-) hepatic enzymes;protection from reactive oxygen species (ROS) by the glutathione (GSH) oxidative defense system, and immune responsiveness (focus on human leukocyte antigen, HLA). Genes encoding the drug metabolizing enzymes, GSH -related enzymes, and HLA proteins are polymorphic, but the role of genetic factors in SMX hypersensitivity remains uncertain. We propose that a comprehensive approach is needed to clarify genetic factors in idiosyncratic reactions. Specific Aim 1 focuses on targeted genotyping of candidate genes (35 candidate genes and 149 polymorphisms) involved in SMX metabolism and toxicity, and in immune response (HLA) using high throughput methods. Genetic analyses will test association between genotype and clinical outcomes, comparing a cohort of HIV/AIDS patients with and without SMX hypersensitivity. To increase the power of clinical genetic association analysis, this study includes a systematic search for novel functional polymorphisms. While most of the candidate genes have been extensively studied with focus on non-synonymous SNPs that change protein structure, regulatory polymorphisms appear to be more prevalent but most remain to be discovered. Specific Aim 2 is to identify novel regulatory SNPs in NAT1 - a highly polymorphic gene - because of its important role in SMX metabolism. The approach relies on measuring allelic ratios in genomic DNA and mRNA of candidate genes in target tissues (150 liver autopsies). Any deviation from equal DNA and mRNA ratios, termed allelic expression imbalance, reveals the presence of cis-acting factors, providing a precise quantitative phenotype. Scanning the gene locus for linked SNPs reveal novel regulatory polymorphisms. Taken together, the proposed study will clarify whether genetic factors contribute to SMX hypersensitivity in HIV/AIDS patients, and identify novel functional polymorphisms in NAT1. This combined approach has promise for the general study of idiosyncratic adverse drug reactions. PUBLIC HEALTH RELEVANCE: This project aims at discovering genetic biomarkers for predicting severe adverse reactions to sulfamethoxazole, widely used in prophylaxis against opportunistic infections associated with AIDS and other immune-compromised conditions. This type of idiosyncratic adverse reactions, likely involving reactive oxygen intermediates and immune response as a causative factor, is associated with many drugs but remains poorly understood. The results of this study will assess the genetic component contributing to adverse sulfamethoxazole reaction, shed light on idiosyncratic drug reactions in general, and guide the selection of optimized therapies for individual patients.